Investigation and comparison of the anti-tumor activities of lactoferrin, α-lactalbumin, and ß-lactoglobulin in A549, HT29, HepG2, and MDA231-LM2 tumor models.

To investigate the anti-tumor activities of lactoferrin, α-lactalbumin, and ß-lactoglobulin, 4 types of human tumor cells (lung tumor cell A549, intestinal epithelial tumor cell HT29, hepatocellular cell HepG2, and breast cancer cell MDA231-LM2) were exposed to 3 proteins, respectively. The effects on cell proliferation, migration, and apoptosis were detected in vitro, and nude mice bearing tumors were administered the 3 proteins in vivo. Results showed that the 3 proteins (20 g/L) inhibited viability and migration, as well as induced apoptosis, in 4 tumor cells to different degrees (compared with the control). In vivo, tumor weights in the HT29 group (0.84 ± 0.22 g vs. control 2.05 ± 0.49 g) and MDA231-LM2 group (1.11 ± 0.25 g vs. control 2.49 ± 0.57 g) were significantly reduced by lactoferrin; tumor weights in the A549 group (1.07 ± 0.19 g vs. control 3.11 ± 0.73 g) and HepG2 group (2.32 ± 0.46 g vs. control 3.50 ± 0.74 g) were significantly reduced by α-lactalbumin. Moreover, the roles of lactoferrin, α-lactalbumin, and ß-lactoglobulin in regulating apoptotic proteins were validated. In summary, lactoferrin, α-lactalbumin, and ß-lactoglobulin were proven to inhibit growth and development of A549, HT29, HepG2, and MDA231-LM2 tumors to different degrees via induction of cell apoptosis.

Mechanical stress contributes to osteoarthritis development through the activation of transforming growth factor beta 1 (TGF-ß1).

OBJECTIVES: The role of mechanical stress and transforming growth factor beta 1 (TGF-ß1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known. METHODS: In this study, TGF-ß1 from osteoclasts and knee joints were analyzed using a co-cultured cell model and an OA rat model, respectively. Five patients with a femoral neck fracture (four female and one male, mean 73.4 years (68 to 79)) were recruited between January 2015 and December 2015. Results showed that TGF-ß1 was significantly upregulated in osteoclasts by cyclic loading in a time- and dose-dependent mode. The osteoclasts were subjected to cyclic loading before being co-cultured with chondrocytes for 24 hours. RESULTS: A significant decrease in the survival rate of co-cultured chondrocytes was found. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay demonstrated that mechanical stress-induced apoptosis occurred significantly in co-cultured chondrocytes but administration of the TGF-ß1 receptor inhibitor, SB-505124, can significantly reverse these effects. Abdominal administration of SB-505124 can attenuate markedly articular cartilage degradation in OA rats. CONCLUSION: Mechanical stress-induced overexpression of TGF-ß1 from osteoclasts is responsible for chondrocyte apoptosis and cartilage degeneration in OA. Administration of a TGF-ß1 inhibitor can inhibit articular cartilage degradation.Cite this article: R-K. Zhang, G-W. Li, C. Zeng, C-X. Lin, L-S. Huang, G-X. Huang, C. Zhao, S-Y. Feng, H. Fang. Mechanical stress contributes to osteoarthritis development through the activation of transforming growth factor beta 1 (TGF-ß1). Bone Joint Res 2018;7:587-594. DOI: 10.1302/2046-3758.711.BJR-2018-0057.R1.

[Using (1)H-nuclear magnetic resonance metabolomics and gene ontology to establish pathological staging model for esophageal cancer patients].

OBJECTIVES: By combining the metabolomics and computational biology, to explore the relationship between metabolic phenotype and pathological stage in esophageal cancer patients, to find the mechanism of metabolic network disturbance and develop a new method for fast preoperative clinical staging. METHODS: A prospective cohort study (from April 2013 to January 2016) was conducted. The preoperative patients from Sichuan Provincial People's Hospital, who were diagnosed with esophageal cancer from May 2013 to April 2014 were included, and their serum samples were collected to detect (1)H-nuclear magnetic resonance (NMR) metabolomics for the purpose of drawing the metabolic fingerprinting in different stages of patients with esophageal cancer. The data were processed with these methods-principal components analysis: partial least squares regression and support vector machine, for the exploration of the enzyme-gene network regulatory mechanism in abnormal esophageal cancer metabolic network regulation and to build the quantitative prediction model of esophageal cancer staging in the end. All data were processed on high-performance computing platforms Matalab. The comparison of data had used Wilcoxon test, variance analysis, χ(2) test and Fisher exact test. RESULTS: Twenty patients with different stages of esophageal cancer were included; and their serum metabolic fingerprinting could differentiate different tumor stages. There were no difference among the five teams in the age (F=1.086, P>0.05), the body mass index (F=1.035, P>0.05), the distance from the incisors to tumor (F=1.078, P>0.05). Among the patients with different TNM stages, there was a significant difference in plasma metabolome. Compared to ⅡB, ⅢA, Ⅳstage patients, increased levels of butanone, ethanol amine, homocysteine, hydroxy acids and estriol, together with decreased levels of glycoprotein, creatine, choline, isobutyricacid, alanine, leucine, valine, were observed inⅠB, ⅡA stage patients. Four metabolic markers (ethanol amine, hydroxy-propionic acid, homocysteine and estriol) were eventually selected. gene ontology analysis showed that 54 enzymes and genes regulated the 4 key metabolic markers. The quantitative prediction model of esophageal cancer staging based on esophageal cancer NMR spectrum were established. Cross-validation results showed that the predicted effect was good (root mean square error=5.3, R(2)=0.47, P=0.036). CONCLUSIONS: The systems biology approaches based on metabolomics and enzyme-gene regulatory network analysis can be used to quantify the metabolic network disturbance of patients with advanced esophageal cancer, and to predict preoperative clinical staging of esophageal cancer patients by plasma NMR metabolomics.

Chondrogenesis of mesenchymal stem cells in a novel hyaluronate-collagen-tricalcium phosphate scaffolds for knee repair.

Scaffolds are expected to play a key role in the induction of chondrogenesis of mesenchymal stem cells (MSCs) for cartilage tissue regeneration. Here, we report the development of a novel tricalcium phosphate-collagen-hyaluronate (TCP-COL-HA) scaffold that can function as a stem cell carrier to induce chondrogenesis and promote cartilage repair, and the investigation of chondroinductive properties of scaffolds containing varying amounts of TCP, COL and HA. TCP-COL-HA scaffolds, as well as TCP-COL scaffolds at two different TCP/COL ratios (50:50 and 25:75), were evaluated for their ability to induce cartilage regeneration from rabbit mesenchymal stem cells (rMSCs) in vitro and in vivo. Chondrogenic differentiation was evaluated by sulphated glycosaminoglycan quantification, collagen type II immunohistochemistry, and qRT-PCR. Mechanical strength was evaluated by the compression test. The results showed that the TCP-COL-HA scaffolds enhanced rMSC chondrogenesis to a greater degree than did the TCP-COL scaffolds; for the latter, the scaffold with the lower TCP/COL ratio (25:75) was superior in terms of promoting rMSC chondrogenesis. Similar results were obtained in an ectopic implantation model in nude mice. In a critical-size rabbit osteochondral defect-repair model, rMSCs seeded on TCP-COL-HA scaffolds showed greater cartilage regeneration and integration into surrounding tissue than the TCP-COL groups, in which cartilage repair was more efficient at the 25:75 than at the 50:50 ratio. These results indicate that the addition of HA and different TCP/COL ratios can affect the chondroinductive capacity of scaffolds, and suggest that the TCP-COL-HA scaffold can serve as an effective cell carrier for cartilage regeneration.

CCL3 serves as a potential plasma biomarker in knee degeneration (osteoarthritis).

OBJECTIVE: To explore the ability of chemokines in plasma to detect the presence of pre-X-rays defined knee degeneration and the extent (burden). METHODS: A total of 181 subjects (75 control subjects, 47 pre-X-KD patients and 50 X-KOA patients) were included and subdivided into three subgroups. Articular cartilage loss in pre-X-KD patients were scored on the basis of the ICRS classification during the arthroscopy or documented on MRI with chondral WORMS. The severity of X-KOA was graded using the Kellgren-Lawrence classification through the posterior-anterior knee X-rays. The concentrations of the inflammatory cytokines and chemokines in plasma were quantified using Luminex microbead-based suspension array (SA) and were cross-validated by enzyme-linked immunosorbent assay (ELISA). RESULTS: CCL3 in plasma showed the highest ability to discriminate pre-X-KD patients from the controls with an AUC of 0.799. At a cutoff value of 0.168 pg/ml, the sensitivity was 70.21%, the specificity was 96.00%, the positive predictive value was 91.67% and the negative predictive value was 83.72%. As to define disease burden, the plasma levels of resistin, IL6, IL8, CCL3 and CCL4 showed significant association with the severity of X-rays defined knee OA, with regard to the KL classification. Moreover, significant elevation of IL6, IL8, CCL3 and CCL4 levels in plasma were observed in severe knee OA patients (KL grade IV) compared with those with pre-X-KD (KL grade 0-I). CONCLUSION: We firstly showed that the plasma CCL3 could be potential serum biomarker for knee OA with the capacity to detect pre-X-rays defined changes and stage the severity of damage in knee.

Multi-lineage MSC differentiation via engineered morphogen fields.

Tissue loss due to oral diseases requires the healing and regeneration of tissues of multiple lineages. While stem cells are native to oral tissues, a current major limitation to regeneration is the ability to direct their lineage-specific differentiation. This work utilizes polymeric scaffold systems with spatiotemporally controlled morphogen cues to develop precise morphogen fields to direct mesenchymal stem cell differentiation. First, a simple three-layer scaffold design was developed that presented two spatially segregated, lineage-specific cues (Dentinogenic TGF-ß1 and Osteogenic BMP4). However, this system resulted in diffuse morphogen fields, as assessed by the in vitro imaging of cell-signaling pathways triggered by the morphogens. Mathematical modeling was then exploited, in combination with incorporation of specific inhibitors (neutralizing antibodies or a small molecule kinase inhibitor) into each morphogen in an opposing spatial pattern as the respective morphogen, to design a five-layer scaffold that was predicted to yield distinct, spatially segregated zones of morphogen signaling. To validate this system, undifferentiated MSCs were uniformly seeded in these scaffold systems, and distinct mineralized tissue differentiation were noted within these morphogen zones. Finally, to demonstrate temporal control over morphogen signaling, latent TGF-ß1 was incorporated into one region of a concentric scaffold design, and laser treatment was used to activate the morphogen on-demand and to induce dentin differentiation solely within that specific spatial zone. This study demonstrates a significant advance in scaffold design to generate precise morphogen fields that can be used to develop in situ models to explore tissue differentiation and may ultimately be useful in engineering multi-lineage tissues in clinical dentistry.

Distribution and pathogen identification of cassava brown leaf spot in China.

Cassava brown leaf spot surveys were conducted in the main cassava plantation areas of China between 2007 and 2012 in order to understand the distribution of the disease. Cassava plants were damaged by the disease to different degrees in most of the survey sites. Samples were collected and seven strains were isolated from lesions. The mycelium-breaking plus black light induction method was applied for sporulation. Microconidia were formed by means of fragmentation on artificial medium plates. When the leaf was stabbed and inoculated with conidia solution, similar symptoms were formed 14 days later. Morphological characteristics of the specimens and conidia were similar to descriptions of Passalora henningsii infection. The internal transcribed spacer (ITS) regions of rDNA were obtained with primer pair ITS1/ITS4 and deposited in GenBank, which differed by three base pairs from that of the P. henningsii isolate (AF284389). The ITS sequences of related species were downloaded from the NCBI database, and phylogenetic analysis showed that the sequences originating from our strains clustered in the same clade as the AF284389 isolate. Biological characteristics were evaluated in two strains from different sites, which indicated that the optimum conditions for mycelia growth were a temperature of 26° to 28°C, carrot agar medium, pH 6, and continuous dark; cassava leaf juice added to malt extract and cassava leaf juice added to potato dextrose agar were the best media for conidia production. The optimal and lethal temperatures for macroconidia germination were 26° to 28°C, and 60°C for 10 min, respectively.

First Report of Rubber Tree Stem Rot Caused by Fusarium oxysporum in China.

Rubber tree (Hevea brasiliensis) is an important crop in tropical regions of China. In October 2013, a new stem rot disease was found on cv. Yunyan77-4 at a rubber tree plantation in Hekou, Yunnan Province. There were about 100 plants, and diseased rubber trees accounted for 30% or less. Initially, brown-punctuate secretion appeared on the stem, which was 5 to 6 cm above the ground. Eventually, the secretion became black and no latex produced from the rubber tree bark. After removing the secretion, the diseased bark was brown putrescence, but the circumambient bark was normal. Upon peeling the surface bark, the inner bark and xylem had brown rot and was musty. The junction between health and disease was undulate. On the two most serious plants, parts of leaves on the crown were yellow, and the root near the diseased stem was dry and puce. The pathogen was isolated and designated HbFO01; the pathogenicity was established by following Koch's postulates. The pathogen was cultivated on a potato dextrose agar (PDA) plate at 28°C for 4 days. Ten plants of rubber tree cv. Yunyan77-4 were selected from a disease-free plantation in Haikou, Hainan Province, and the stem diameter was about 7 cm. The bark of five plants was peeled, and one mycelium disk with a diameter of 1 cm was inserted into the cut and covered again with the bark. The other five plants were treated with agar disks as controls. The inoculation site was kept moist for 2 days, and then the mycelium and agar disk were removed. On eighth day, symptoms similar to the original stem lesions were observed on stems of inoculated plants, while only scars formed on stems of control plants. The pathogen was re-isolated from the lesions of inoculated plants. On PDA plates, the pathogen colony was circular and white with tidy edges and rich aerial hyphae. Microscopic examination showed microconidia and chlamydospores were produced abundantly on PDA medium. The falciform macroconidia were only produced on lesions and were slightly curved, with a curved apical cell and foot shaped to pointed basal cell, usually 3-septate, 16.2 to 24.2 × 3.2 to 4.0 µm. Microconidia were produced in false heads, oval, 0-septate, 6.2 to 8.2 × 3.3 to 3.8 µm, and the phialide was cylindrical. Chlamydospores were oval, 6.4 to 7.2 × 3.1 to 3.8 µm, alone produced in hypha. Morphological characteristics of the specimen were similar to the descriptions for Fusarium oxysporum (2). Genomic DNA of this isolate was extracted with a CTAB protocol (4) from mycelium and used as a template for amplification of the internal transcribed spacer (ITS) region of rDNA with primer pair ITS1/ITS4 (1). The full length of this sequence is 503 nt (GenBank Accession No. KJ009335), which exactly matched several sequences (e.g., JF807394.1, JX897002.1, and HQ451888.1) of F. oxysporum. Williams and Liu had listed F. oxysporum as the economically important pathogen of Hevea in Asia (3), while this is, to our knowledge, the first report of stem rot caused by F. oxysporum on rubber tree in China. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual, 2006. (3) T. H. Williams and P. S. W. Liu. A host list of plant diseases in Sabah, Malaysia, 1976. (4) J. R. Xu et al. Genetics 143:175, 1996.

First Report of Alternaria heveae Causing Black Leaf Spot of Rubber Tree in China.

Rubber tree (Hevea brasiliensis Muell. Arg.) is an important industrial crop of tropical areas for natural rubber production. In October 2013, foliar spots (0.1 to 0.4 mm in diameter), black surrounded by a yellow halo, and with lesions slightly sunken were observed on the rubber tree leaf in a growing area in Heikou County of Yunnan Province. Lesion tissues removed from the border between symptomatic and healthy tissue were surface sterilized in 75% ethanol and air-dried, plated on PDA plates, and incubated at 28°C with alternating day/night cycles of light. The pathogen was observed growing out of many of the leaf pieces, and produced abundant conidia. Colonies 6.1 cm in diameter developed on potato carrot agar (PCA) after 7 days, with well-defined concentric rings of growth. Colonies on PCA were composed of fine, dark, radiating, surface and subsurface hyphae. Conidia produced in PCA culture were mostly solitary or in short chains of 2 to 5 spores, long ovoid to clavate, and light brown, 40 to 81.25 × 8 to 20 µm (200 colonies were measured), with 3 to 6 transverse septa and 0 to 2 longitudinal or oblique septa. Morphological characteristics were similar to those described for Alternaria heveae (3,4). A disease of rubber tree caused by Alternaria sp. had been reported in Mexico in 1947 (2). DNA of Ah01HK13 isolate was extracted for PCR and sequencing of the ITS region with ITS1 and ITS4 primers was completed. From the BLAST analysis, the sequence of Ah01HK13 (GenBank Accession No. KF953884), had 97% similarity to A. dauci, 96% identical to A. macrospora (AY154701.1 and DQ156342.1, respectively), indicating the pathogen belonged to Alternaria genus. According to morphological characteristics, this pathogen was identified as A. heveae. Pathogenicity of representative isolate, Ah01HK13 was confirmed using a field rubber tree inoculation method. Three rubber plants (the clone of rubber tree Yunyan77-4) were grown to the copper-colored leaf stage and inoculated by spraying spore suspension (concentration = 104 conidia/ml) to the copper-colored leaves until drops were equally distributed on it using manual pressure sprayer. Three rubber plants sprayed with sterile distilled water were used as controls. After inoculation, the plants were covered with plastic bags. The plastic bags were removed after 2 days post-inoculation (dpi) and monitored daily for symptom development (1). The experiment was repeated three times. The typical 0.1 to 0.4 mm black leaf spots were observed 7 dpi. No symptoms were observed on control plants. A fungus with the same colony and conidial morphology as A. heveae were re-isolated from leaf lesions on inoculated rubber plants, but not from asymptomatic leaves of control plants, fulfilling Koch's postulates. Based on these results, the disease was identified as black spot of rubber tree caused by A. heveae. To our knowledge, this is the first report of A. heveae on rubber tree in China. References: (1) Z. Y. Cai et al. Microbiol Res. 168:340, 2013. (2) W. J. Martin. Plant Dis. Rep. 31:155, 1947. (3) E. G. Simmons. Mycotaxon 50:262, 1994. (4) T. Y. Zhang. Page 111 in: Flora Fungorum Sinicorum: Alternaria, Science Press, Beijing, 2003.

Strongly interacting photons in asymmetric quantum well via resonant tunneling.

We propose an asymmetric quantum well structure to realize strong interaction between two slow optical pulses. The essential idea is the combination of the advantages of inverted-Y type scheme and resonant tunneling. We analytically demonstrate that giant cross-Kerr nonlinearity can be achieved with vanishing absorptions. Owing to resonant tunneling, the contributions of the probe and signal cross-Kerr nonlinearities to total nonlinear phase shift vary from destructive to constrictive, leading to nonlinear phase shift on order of π at low light level. In this structure, the scheme is inherent symmetric for the probe and signal pulses. Consequently, the condition of group velocity matching can be fulfilled with appropriate initial electron distribution.

First Report of Phytophthora palmivora Causing Root Rot of Cassava in China.

Cassava (Manihot esculenta) is an economically important crop grown widely in South China. Seventy percent of the cassava grown is used for starch and ethanol production and it has become the foundation of local food and bioenergy systems. In November 2010, a new root rot disease was found on cv. HuaNan205 from a cassava plantation in Danzhou, Hainan Province. Disease occurred on 30% or less of the plants. Initially, the upper leaves wilted at noon and recovered in the evening. Eventually, infected plants no longer recovered and the whole plant wilted and died. Root rot symptoms consisting of irregular brown patches occurred on the tuberous roots. Symptomatic root rot tissue was cut into 1-cm pieces, washed in distilled water, and soaked in a solution of 1% sodium hypochlorite for 3 min. A subsection was cut from each sterilized piece, placed on a plate of V8 agar medium, and incubated at 28°C for 7 days. Pathogenicity was established by following Koch's postulates. In July 2011, 10 plants of cassava cv. HuaNan205 were selected from a disease-free plantation in Danzhou. The pathogen was cultivated on V8 agar at 28°C for 14 days. Four holes were established 15 cm from the base of the cassava plants. Five plants were inoculated with 100 mL of the mycelial suspension in each of the four spots and covered by soil. The other five plants were treated with sterile water as control. Plants were maintained for 4 months. All five of the inoculated plants wilted and two died, while the control plants grew normally. Symptoms similar to the original root lesions were observed on tuberous roots of inoculated plants, while only scars formed on tuberous roots of control plants. The pathogen was reisolated from the lesions of inoculated plants. Microscopic examination showed the sporangia as papillate and ovoid with the widest part close to the base. They were easily washed off and each detached sporangium contained a short pedicel 1.2 to 6.9 µm long, average 2.9 µm. Chlamydospores were readily observed on diseased roots and observed in pure cultures on V8 agar. Morphological characteristics of the specimen were similar to the descriptions for Phytophthora palmivora (2). Genomic DNA of this isolate was extracted with a cetyltrimethylammoniumbromide protocol (3) from mycelium and used as a template for amplification of the internal transcribed spacer (ITS) region of rDNA with primer pair ITS1/ITS4 (1). The sequence (GenBank Accession No. HE580279) exactly matched several sequences (e.g., GenBank Accession Nos. HQ237481.1, AY745750, and AY745751) of P. palmivora. To our knowledge, this is the first report of root rot caused by P. palmivora on cassava in China. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) J. R. Xu et al. Genetics 143:175, 1996.

First Report of Corynespora cassiicola Causing Leaf Spot of Cassava in China.

Cassava (Manihot esculenta) is an important economic crop in the tropical area of China. During a survey of diseases in July and September of 2009, leaf spots were observed on cassava plants at three separate plantations in Guangxi (Yunfu and Wuming) and Hainan (Baisha) provinces. Circular or irregular-shaped leaf spots were present on more than one-third of the plants. Spots were dark brown or had white papery centers delimited by dark brown rims and surrounded by a yellow halo. Usually, the main vein or small veinlets adjacent to the spots were dark. Some defoliation of plants was evident at the Wuming location. A fungus was isolated from symptomatic leaves from each of the three locations and designated CCCGX01, CCCGX02, and CCCHN01. Single-spore cultures of these isolates were incubated on potato dextrose agar (PDA) for 7 days with a 12-h light/dark cycle at a temperature of 28 ± 1°C. Conidiophores were straight to slightly curved, unbranched, and pale to light brown. Conidia were formed singly or in chains, obclavate to cylindrical, straight or curved, subhyaline-to-pale olivaceous brown, 19.6 to 150.3 µm long and 5.5 to 10.7 µm wide at the base, with 4 to 13 pseudosepta. Morphological characteristics of the specimen and their conidia were similar to the descriptions for Corynespora cassiicola (2). The isolate CCCGX01 was selected as a representative for molecular identification. Genomic DNA was extracted by the cetyltrimethylammoniumbromide protocol (3) from mycelia and used as a template for amplification of the internal transcribed spacer (ITS) region of rDNA with primer pair ITS1/ITS4. The sequence (GenBank Accession No. GU138988) exactly matched several sequences (e.g., GenBank Accession Nos. FJ852715, EF198117, and AY238606) of C. cassiicola (1). Young, healthy, and fully expanded green leaves of cassava cv. SC205 were surface sterilized. Ten leaves were inoculated with 10-µl drops of 104 ml suspension of conidia and five leaves were inoculated with the same volume of sterile water to serve as controls. After inoculation, leaves were placed in a dew and dark chamber for 36 h at 25°C and subsequently transferred to the light for 5 days. All inoculated leaves with isolates showed symptoms similar to those observed in natural conditions, whereas the controls remained symptom free. The morphological characteristics of reisolated conidia that formed on the diseased parts were identical with the nature isolates. To our knowledge, this is the first report of leaf spot caused by C. cassiicola on cassava in China. References: (1) L. J. Dixon et al. Phytopathology 99:1015, 2009. (2) M. B. Ellis et al. Corynespora cassiicola. No. 303 in: CMI Description of Pathogenic Fungi and Bacteria. Commonwealth Mycological Institute, Kew, UK 1971. (3) J. R. Xu et al. Genetics 143:175, 1996.

First Report of Leaf Spot Caused by Bipolaris setariae on Cassava in China.

Cassava (Manihot esculenta Crantz) is an important food crop in tropical regions of China. Seventy percent of the cassava output is used for starch and ethanol production and it has become the base of food and bioenergy industries. In July 2009, a new leaf spot disease was found on cv. HuaNan205 from a cassava plantation in Danzhou, Hainan Province. Disease occurred on 50% or less of the plants. Initial symptoms were elliptical, chlorotic, and water-immersion lesions of 2 to 4 mm in diameter. These lesions became dry and yellow due to the progress of the disease. A brown halo was around the lesions, and in wet conditions, a dark gray mildew often appeared in the middle of the lesion. Diseased leaves turned yellow and the plants eventually became defoliated. The pathogen was isolated and pathogenicity was established by following Koch's postulates. Young, healthy, and fully expanded green leaves of Cassava cv. HuaNan205 were surface sterilized and then inoculated by spraying them with a suspension of conidia (1 × 105 conidia per ml) of the isolate. Sterile water was used as a control. The leaves were kept in a humid chamber at 28°C for 4 days, at which time similar symptoms to those described above were observed on the leaves. The pathogen was reisolated from inoculated leaves. Microscopic examination showed the conidiophores were fasciculate and brown, septate and straight, and the basal cell was enlarged and hemispherical. Well-developed conidia were long-obclavate, obtuse at both ends, straight, brown, with five to eight transverse septa, and measured 49.7 to 117.1 × 13.3 to 17.2 µm. Genomic DNA of this isolate was extracted with a cetyltrimethylammoniumbromide protocol, and amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA was performed with procedures outlined by Cooke et al. (2). The sequence of the region was deposited in GenBank (Accession No. GU290228). Comparison of the sequences available in the GenBank database revealed that the current ITS sequence differs by three base pairs from two Bipolaris setariae isolates (EF452444 and FJ606786). Morphological identification and sequence analysis of ITS rDNA showed that the pathogen was B. setariae. B. setariae is one of the most important pathogens of lawn grass, gramineous crops, and other plants (1,3). However, no leaf spot disease caused by B. setariae has been recorded previously on cassava in China or elsewhere. References: (1) P. Busey. Crop Sci. 43:1899, 2003. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) H. D. Wells and W. W. Hanna. Phytopathology 78:1179, 1988.

[p16 and cyclinD1 protein expression and p16 gene mutation in primary human hepatocellular carcinoma].

OBJECTIVE: To elucidate the relationship between expression of p16 and cyclinD1 proteins and evaluate the role of p16 gene exon 2 mutation in hepatocellular carcinogenesis. METHODS: Streptavidin-peroxidase conjugated method(SP) was used to detect expressions of p16 and cyclinD1 proteins in 44 cases of hepatocellular carcinoma(HCC), and polymerase chain reaction single-strand conformation polymorphism analysis(PCR-SSCP) to detect p16 gene mutation in exon 2 in 12 cases of HCC and liver tissues adjacent to HCC. RESULTS: Expression rate and positive signal intensity of p16 protein in HCC were significantly lower(P < 0.01) and those of cyclinD1 protein in HCC were significantly higher (P < 0.05) than those in pericarcinomatous tissues. Of 12 fresh HCC tissues, p16 gene mutation in exon 2 was found in 2 cases, whereas that was not found in pericarcinomatous tissues. CONCLUSIONS: 1. Inactivation or/ and deletion of p16 protein may be one of important reasons which result in proliferation unbalance of cells. 2. p16 gene mutation in exon 2 presents in HCC, but it does not frequently occur in Chinese hepatocarcinogenesis. 3. p16 gene abnormality and cyclinD1 over expression may coact in HCC.

[Modified breast reconstruction by latissimus dorsi musculocutaneous flap].

OBJECTIVE: To investigate the effect of breast reconstruction with latissimus dorsi musculocutaneous flap. METHODS: Since 1994, 60 cases were performed breast reconstruction with latissimus dorsi musculocutaneous flap with fat tissue nourished by thoracodorsal artery according to the shape and volume of the normal breast on the other side. All of cases were followed up for 3 months to 5 years. RESULTS: Among the 60 cases, excellent effect was obtained in 41 cases (68.3%), good effect in 16 cases (26.7%), unsatisfactory in 3 cases (5.0%). CONCLUSION: Modified latissimus dorsi musculocutaneous flap to reconstruct breast overcome the shortcoming of volume deficiency of traditional latissimus dorsi in breast reconstruction, and it is a safe and easy-manipulated surgical operation.